Project Summary: Our long-term objective is to improve our understanding of human lung development and disease using iPSC directed differentiations to access cells from the same human being, at various stages of lung development. Basal cells are epithelial stem/progenitor cells of the airway. They exhibit self-renewal and multi-lineage differentiation in vitro and in vivo and can reconstitute the injured airway epithelium. Deriving functional basal cells from induced pluripotent stem cells (iPSCs) would have broad ranging applications for disease modeling, drug development, and potential cell-based therapies. The goal of this proposal is to (1) identify for the first time the signaling pathways that control human airway BC specification, (2) determine the in vitro conditions that support self-renewal and expansion of iPSC-derived BCs, (3) compare the functional and transcriptional profile of these cells to bone-fide adult BCs, and (4) employ these engineered cells to model the monogenic airway disease cystic fibrosis (CF). The central hypothesis is that precise step-wise modulation of developmental pathways can be used to dissect the mechanisms of human basal cell specification, maturation and differentiation and to model airway diseases. We present preliminary data demonstrating the feasibility of deriving BCs from human iPSCs, including functional and single-cell RNA- Seq (scRNA-Seq) characterizations. We introduce for the first time a novel human iPSC reporter line to track and purify putative BCs. Based on mouse studies and our preliminary human data we hypothesize that FGF and Hippo signaling play key roles in human BC specification and subsequent maintenance/amplification of the progenitor pool. We propose a detailed comparison of iPSC-derived stem cells to their in-vivo counterparts to establish their similarity but also harness the information to improve the differentiation protocols for iPSCs.